G-quadruplex (G4) and i-motif (iM) structures represent a new class of molecular targets for antitumor therapies. These structures occur in G/C-rich DNA in proximity to transcriptional start sites, particularly in oncogenes. Formation of G4/iM structures in DNA modulates transcription of genes. Their globular nature allows for our long-term goal of specific targeting by small molecules, and there is sufficient variation in G4/iM structures across promoters to allow for focused efforts on specific genes. Previously, these structures have been studied in vitro, primarily using single-stranded DNA. However, due to their structural polymorphism, little remains known about (i) G4/iM formation in vivo, (ii) how endogenous cellular conditions contribute to overcoming energy barriers to their formation, and (iii) the most physiologically relevant structures to target. The objective of our proposal is to eliminate this fundamental gap in knowledge of precise G4/iM structures under in vivo conditions in two cancer-related genes - MYC and VEGF - that have been well-studied under in vitro conditions. Our central hypothesis is that the combination of endogenous cellular biophysical and chemical stressors will result in novel G4/iM structures that have yet to be detected, and that these novel structures are of critical biological relevance for understanding regulation of expression of these genes and ultimately for drug design. This hypothesis is supported by many literature examples of structural differences between those observed under simple in vitro conditions and those observed with added biologically relevant stressors. Our expected outcome is that in vivo stressors will result in significantly different structures as compared to simple in vitro conditions, and will provide novel in vivo targets for understanding gene regulation and antitumor drug development. We will test our central hypothesis with the following two specific aims: 1) Quantify the kinetic and thermodynamic properties, nucleotide interactions, and underlying equilibrating G4s/iM structures in the presence of 5-hydroxymethylated cytosines and 2) Determine structural equilibria and protein-DNA interactions of nucleolin and hnRNP K with the G4s and iMs, respectively, within the MYC and VEGF promoters. In order to accomplish these aims, we will use a combination of G4/iM techniques to probe the higher order DNA structures in both single-stranded and supercoiled DNA. The research proposed here is innovative in that we will study G4/iM structures under compounded conditions found in vivo, which for the first time will include the incorporation of 5-hydroxymethylated cytidines. While MYC and VEGF have importance for understanding and treatment of cancer, we anticipate that our studies will have a positive impact on other high-value genes important in biomedicine. Additionally, in support of the AREA program goals, this work provides hands-on learning for students at the University of Mississippi, who will study fundamental questions regarding higher-order DNA dynamics.